The goal of this proposal is to advance our understanding of the mechanism of action and function of the matrix metalloproteinase-20 (enamelysin) during enamel biomineralization. Recent findings on the abnormal enamel formation in transgenic enamelysin-deficient mice have highlighted the critical function of enamelysin during enamel development. The study on the stepwise processing of the enamel extracellular matrix components is essential for the understanding of pathological dental enamel formation and is one of the critical steps towards the development of enamel-inspired biomimetic materials. Our general hypothesis is that MMP-20 cleaves specific domains: a) within amelogenin to alter the assembly of its proteolytic products and their interactions with apatite crystals as well as the structural organization of the extracellular framework, b) within enamelin and ameloblastin to generate polypeptides with defined physiological function such as control of crystal nucleation and growth. The following specific aims are proposed to examine the above hypothesis: I) To examine peptide bond specificity of MMP-20 by: a) using commercially available polypeptide substrates, b) systematically determining MMP-20 cleavage site motifs at both the N-terminal (P1-Pn) and C-terminal (P1'-Pn') using mixture-based oriented peptide libraries.Il) To determine cleavage sites on recombinant amelogenin rp172 and rp148 by MMP-20 in solution as well as adsorbed on isolated enamel crystals. Ill) To investigate the effect of MMP-20 action on the assembly and disassembly of amelogenin nanospheres in solution. IV) To investigate the effect of MMP-20 action on the structural organization of the amelogenin matrix in a "gel-like" state using atomic force microscopy, SEM, and dynamic light scattering. V) To determine cleavage sites on recombinant ameloblastin by MMP-20 in solution as well as adsorbed onto isolated enamel crystals. VI) To examine the action of MMP-20 on synthetic peptides derived from potential cleavage sites on enamelin proteins. In summary: The proposed in vitro studies will be complementary to current research on amelogenesis when MMP-20, amelogenin, ameloblastin, and enamelin null and transgenic mice strategies are applied. The knowledge gained from our proposed experiments will provide a solid base for interpretation of the in vivo animal model studies. The scientific chemical principles gained from the proposed in vitro studies will have a great impact on the field of enamel biomineralization, matrix metalloproteinases, tooth development, and our understanding of pathological enamel formation. In addition, these studies will contribute to the basic knowledge required for the design and development of novel biomaterials with potential future application in clinical dentistry and other areas of biomedical and biomaterial technology.